2007 Annual Report 1a.Objectives (from AD-416) 1) Identify commensal sources of tetracycline resistance genes;. 2)Evaluate bacteriophage as agents of gene transfer;. 3)Identify protozoal factors that affect pathogen virulence in the rumen; and. 4)Identify dietary strategies to limit acidosis and pathogen reservoirs. 1b.Approach (from AD-416) Isolate commensal bacteria from swine that share niches and exchange genes with Campylobacter. Classify Campylobacter strains for antibiotic susceptibility and amplify and sequence tet genes. Add carbadox to stimulate phage-induced tylosin resistance gene transfer in Brachyspira. Assay degree of phage induction and gene transfer. Harvest protozoa from rumen contents and determine associated bacterial populations using ARISA and BLAST. Culture single species of protozoa and allow them to feed upon specific bacteria tagged with fluorescence. Examine protozoa for uptake and sequestration of tagged bacteria. Identify compounds to defaunate the rumen and verify reservoir hypothesis by loss of bacterial pathogens in ruminants. 3.Progress Report As a result of personnel loss(es) on this project, modifications have been made with regard to the timing, personnel assignments, and nature of specific milestones as described above and below. Approximately 170 strains of anaerobic bacteria were selectively isolated from the cecal mucosal tissues of turkeys, identified by molecular fingerprinting, and cryo-preserved. These poultry bacteria were selected based on their potential to interact either physiologically or genetically with Campylobacter species. Thirty Megasphaera elsdenii strains were isolated from the ceca of swine from Nebraska and Iowa. Due to its habitat and ecological niche, Megasphaera elsdenii is a commensal bacterium that is likely to interact with Campylobacter in the intestinal tract. Additional commensal bacteria will be isolated from swine mucosal surfaces by end of FY 2007. These strains will be used for investigating mechanisms of antibiotic resistance gene transfer, for applications as probiotic strains to reduce antibiotic resistant bacterial populations in swine, and for examining their genetic and physiological interactions with Campylobacter and other pathogenic bacteria. A previously published assay for monitoring horizontal gene transfer between bacteria in vitro (filter mating assay) was adopted for use in assessing antibiotic resistance gene transfer among pathogenic and non-pathogenic bacteria from the swine intestinal tract. This assay was used successfully to demonstrate the mobility (transfer) of mosaic tetracycline resistance genes between strains of the commensal anaerobe Megasphaera elsdenii. The genes are transferred at a very high frequency by a yet-to-be-identified mechanism which is currently under scrutiny. Megasphaera elsdenii is one of the most highly tetracycline-resistant bacteria colonizing intestinal surfaces of swine, and strains are also multiply drug resistant. The mosaic tetracycline resistance genes are presumed to be the basis of the high tetracycline resistance. DNA sequences of these mosaic tet genes indicate that portions of the genes are identical to those of the swine pathogen Streptococcus suis and the foodborne pathogens Campylobacter and Enterococcus faecalis. The sequence matches suggest that intergeneric genetic exchanges between Megasphaera elsdenii and the pathogens are feasible and these exchanges will be tested as part of the effort to identify commensal bacteria interacting with Campylobacter and other pathogens. 4.Accomplishments None. 5.Significant Activities that Support Special Target Populations None. 6.Technology Transfer Number of web sites managed 1 Number of non-peer reviewed presentations and proceedings 3 Review Publications Matson, E.G., Zuerner, R.L., Stanton, T.B. 2007. Induction and transcription of VSH-1, a prophage-like, gene transfer agent of Brachyspira hyodysenteriae. Anaerobe. 13(2):89-97. Duncan, S.H., Aminov, R.I., Scott, K.P., Louis, P., Stanton, T.B., Flint, H.J. 2006. Proposal of Roseburia faecis sp. nov., Roseburia hominis sp. nov. and Roseburia inulinivorans sp. nov., based on isolates from human faeces. International Journal of Systematic and Evolutionary Microbiology. 56(10):2437-41. Stanton, T.B. 2007. Prophage-like gene transfer agents-novel mechanisms of gene exchange for Methanococcus, Desulfovibrio, Brachyspira, and Rhodobacter species. Anaerobe. 13(2):43-49.